Mutant Taq Polymerase for Increased Salt Concentration or Body Fluids

ABSTRACT

The invention includes a mutant Taq polymerase, which can effectively amplify a target sequence under conditions of salt concentration(s) similar to body fluids, including blood, serum or plasma preserved with sodium citrate. The mutant Taq polymerase, or a biologically active fragment thereof, has one or more substitutions differing from the wild type as shown in Table I.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 28, 2020, isnamed Abclonal-Salt_SL.txt and is 2,582,368 bytes in size.

BACKGROUND

During the polymerase chain reaction (PCR) for DNA amplification thetarget:primer:polymerase mixture is subjected to successive rounds ofheating at different temperatures to facilitate target DNA strandde-annealing (usually performed at about 90-99° C.), primer:target DNAstrand annealing (usually performed at about 40-70° C.), and DNApolymerase-mediated primer elongation (usually performed at about 50-72°C.) to create new complementary amplicon strands. The reaction mayinclude as many as 25-45 rounds of cycling to yield sufficientamplification.

PCR is usually conducted using thermostable DNA polymerases that canwithstand the high temperatures associated with de-annealing withoutsuffering inactivation by heat-induced protein denaturation.

A common problem with diagnostic and forensic PCR is false-negativereactions or low sensitivity caused by inhibitory substances, includingblood samples, that interfere with PCR by affecting the DNA polymerase.See U.S. Pat. No. 8,470,563 (incorporated by reference) and referencescited therein. High salt concentrations in a reaction mixture can alsointerfere with PCR, by affecting the DNA polymerase.

High salt concentrations are often present in preserved blood, plasma,serum and other body fluids and derivatives, because sodium citrate isoften added as a preservative. Magnesium ions are also often present inconcentrations high enough to affect activity of DNA polymerase inpreserved blood, plasma and serum.

Accordingly, a thermostable DNA polymerase which can promote fast andefficient PCR amplification for whole blood samples, plasma, serum andother body fluids and other derivatives, including such samplespreserved with sodium citrate or otherwise having a high saltconcentration, or a high magnesium concentration, is clearly needed.

SUMMARY

The invention includes mutant Taq polymerase with one or more pointmutations differing from the wild type (SEQID NO: 4 shows the wild typeamino acid sequence with a C-terminal His tag; SEQID NO: 5 shows thewild type nucleotide sequence), having one or more of the amino acidpoint substitution shown below in Table I (at one or more of thefollowing positions):

TABLE I   Mutations and Corresponding Sequence ID Numbers E39K SEQ IDNOS: 6-7, E39K/E189K SEQ ID NOS: 8-9, E39K/E230K SEQ ID NOS: 10-11,E39K/D320R SEQ ID NOS: 12-13, E39K/E507K SEQ ID NOS: 14-15, E39K/E520KSEQ ID NOS: 16-17, E39K/E537K SEQ ID NOS: 18-19, E39K/D578R SEQ ID NOS:20-21, E39K/D732R SEQ ID NOS: 22-23, E39K/E742K SEQ ID NOS: 24-25 E76KSEQ ID NOS: 26-27, D91R SEQ ID NOS: 28-29, E101K SEQ ID NOS: 30-31,D104R SEQ ID NOS: 32-33, L111S SEQ ID NOS: 34-35, T186I SEQ ID NOS:36-37, E189K SEQ ID NOS: 38-39, E189D SEQ ID NOS: 40-41, E189L SEQ IDNOS: 42-43, E189M SEQ ID NOS: 44- 45, E189P SEQ ID NOS: 46-47, E189Q SEQID NOS: 48-49, E189R SEQ ID NOS: 50-51, E189T SEQ ID NOS: 52-53, E189YSEQ ID NOS: 54-55, E189K/E230K/E507K SEQ ID NOS: 56-57,E189K/E230K/E520K SEQ ID NOS: 58-59, E189K/E507K/E520K SEQ ID NOS:60-61, E189K/E230K SEQ ID NOS: 62-63, E189K/E507K SEQ ID NOS: 64-65,E189K/E520K SEQ ID NOS: 66-67, E189K/E537K SEQ ID NOS: 68-69,E189K/D578R SEQ ID NOS: 70-71, E189K/D732R SEQ ID NOS: 72-73,E189K/E742K SEQ ID NOS: 74-75, E189K/E230K/E537K SEQ ID NOS: 76-77,E189K/E507K/E537K SEQ ID NOS: 78-79, E189K/E520K/E537K SEQ ID NOS:80-81, E189K/E230K/D578R SEQ ID NOS: 82-83, E189K/E507K/D578R SEQ IDNOS: 84-85, E189K/E520K/D578R SEQ ID NOS: 86-87, E189K/E537K/D578R SEQID NOS: 88-89, E189K/E230K/D732R SEQ ID NOS: 90-91, E189K/E507K/D732RSEQ ID NOS: 92-93, E189K/E520K/D732R SEQ ID NOS: 94-95,E189K/E537K/D732R SEQ ID NOS: 96-97, E189K/D578R/D732R SEQ ID NOS:98-99, E189K/E230K/E742K SEQ ID NOS: 100-101, E189K/E507K/E742K SEQ IDNOS: 102-103, E189K/E520K/E742K SEQ ID NOS: 104- 105, E189K/E537K/E742KSEQ ID NOS: 106-107, E189K/D578R/E742K SEQ ID NOS: 108-109,E189K/D732R/E742K SEQ ID NOS: 110-111 E201K SEQ ID NOS: 112-113, E209KSEQ ID NOS: 114-115, E215K SEQ ID NOS: 116-117, K219E SEQ ID NOS:118-119, L221S SEQ ID NOS: 120-121, D222R SEQ ID NOS: 122-123, E230K SEQID NOS: 124-125, E230M SEQ ID NOS: 126-127, E230S SEQ ID NOS: 128-129,E230T SEQ ID NOS: 130-131, E230V SEQ ID NOS: 132-133, E230W SEQ ID NOS:134-135, E230K/E507K SEQ ID NOS: 136-137, E230K/E520K SEQ ID NOS:138-139, E230K/E537K SEQ ID NOS: 140- 141, E230K/D578R SEQ ID NOS:142-143, E230K/D732R SEQ ID NOS: 144-145, E230K/E742K SEQ ID NOS:146-147, E230K/E507K/E520K SEQ ID NOS: 148-149, E230K/E507K/E537K SEQ IDNOS: 150-151, E230K/E520K/E537K SEQ ID NOS: 152-153, E230K/E507K/D578RSEQ ID NOS: 154-155, E230K/E520K/D578R SEQ ID NOS: 156-157,E230K/E537K/D578R SEQ ID NOS: 158-159, E230K/E507K/D732R SEQ ID NOS:160-161, E230K/E520K/D732R SEQ ID NOS: 162-163, E230K/E537K/D732R SEQ IDNOS: 164-165, E230K/D578R/D732R SEQ ID NOS: 166-167, E230K/E507K/E742KSEQ ID NOS: 168-169, E230K/E520K/E742K SEQ ID NOS: 170-171,E230K/E537K/E742K SEQ ID NOS: 172-173, E230K/D578R/E742K SEQ ID NOS:174-175, E230K/D732R/E742K SEQ ID NOS: 176-177, L233S SEQ ID NOS:178-179, V256S SEQ ID NOS: 180-181, K260E SEQ ID NOS: 182-183, R261D SEQID NOS: 184-185, E288K SEQ ID NOS: 186-187, E303K SEQ ID NOS: 188-189,F309A SEQ ID NOS: 190-191, E315K SEQ ID NOS: 192-193, D320R SEQ ID NOS:194-195, R328D SEQ ID NOS: 196-197, G330P SEQ ID NOS: 198-199, P336G SEQID NOS: 200-201, E337K SEQ ID NOS: 202-203, P338G SEQ ID NOS: 204-205,L342S SEQ ID NOS: 206-207, L351S SEQ ID NOS: 208-209, L365S SEQ ID NOS:210-211, G366P SEQ ID NOS: 212-213, P368G SEQ ID NOS: 214-215, P373G SEQID NOS: 216-217, D381R SEQ ID NOS: 218-219, N384R SEQ ID NOS: 220-221,D452R SEQ ID NOS: 222-223, Y455A SEQ ID NOS: 224-225, N485R SEQ ID NOS:226-227, E507K SEQ ID NOS: 228-229, E507A SEQ ID NOS: 230-231, E507G SEQID NOS: 232-233, E507L SEQ ID NOS: 234-235, E507T SEQ ID NOS: 236-237,E507K/E520K SEQ ID NOS: 238-239, E507K/E537K SEQ ID NOS: 240-241,E507K/D578R SEQ ID NOS: 242- 243, E507K/D732R SEQ ID NOS: 244-245,E507K/E742K SEQ ID NOS: 246-247, E507K/E520K/E537K SEQ ID NOS: 248-249,E507K/E520K/D578R SEQ ID NOS: 250-251, E507K/E537K/D578R SEQ ID NOS:252-253, E507K/E520K/D732R SEQ ID NOS: 254-255, E507K/E537K/D732R SEQ IDNOS: 256-257, E507K/D578R/D732R SEQ ID NOS: 258-259, E507K/E520K/E742KSEQ ID NOS: 260-261, E507K/E537K/E742K SEQ ID NOS: 262-263,E507K/D578R/E742K SEQ ID NOS: 264-265, E507K/D732R/E742K SEQ ID NOS:266-267, E520K SEQ ID NOS: 268-269, E520F SEQ ID NOS: 270-271, E520G SEQID NOS: 272-273, E520H SEQ ID NOS: 274-275, E520I SEQ ID NOS: 276-277,E520N SEQ ID NOS: 278-279, E520Q SEQ ID NOS: 280-281, E520R SEQ ID NOS:282-283, E520S SEQ ID NOS: 284-285, E520T SEQ ID NOS: 286-287, E520Y SEQID NOS: 288-289, E520K/E537K SEQ ID NOS: 290-291, E520K/D578R SEQ IDNOS: 292-293, E520K/D732R SEQ ID NOS: 294- 295, E520K/E742K SEQ ID NOS:296-297, E520K/E537K/D578R SEQ ID NOS: 298-299, E520K/E537K/D732R SEQ IDNOS: 300-301, E520K/D578R/D732R SEQ ID NOS: 302-303, E520K/E537K/E742KSEQ ID NOS: 304-305, E520K/D578R/E742K SEQ ID NOS: 306-307,E520K/D732R/E742K SEQ ID NOS: 308-309, A521F SEQ ID NOS: 310-311, Q534RSEQ ID NOS: 312-313, E537K SEQ ID NOS: 314-315, E537A SEQ ID NOS:316-317, E537F SEQ ID NOS: 318-319, E537G SEQ ID NOS: 320-321, E537H SEQID NOS: 322-323, E537L SEQ ID NOS: 324-325, E537N SEQ ID NOS: 326-327,E537P SEQ ID NOS: 328-329, E537Q SEQ ID NOS: 330-331, E537R SEQ ID NOS:332-333, E537S SEQ ID NOS: 334-335, E537T SEQ ID NOS: 336-337, E537V SEQID NOS: 338-339, E537Y SEQ ID NOS: 340-341, E537K/D578R SEQ ID NOS:342-343, E537K/D732R SEQ ID NOS: 344-345, E537K/E742K SEQ ID NOS: 346-347, E537K/D578R/D732R SEQ ID NOS: 348-349, E537K/D578R/E742K SEQ IDNOS: 350-351, E537K/D732R/E742K SEQ ID NOS: 352-353, L541S SEQ ID NOS:354-355, I546S SEQ ID NOS: 356-357, D547R SEQ ID NOS: 358-359, P550G SEQID NOS: 360-361, D551R SEQ ID NOS: 362-363, L552S SEQ ID NOS: 364-365,I553S SEQ ID NOS: 366-367, P555G SEQ ID NOS: 368-369, N565R SEQ ID NOS:370-371, D578R SEQ ID NOS: 372-373, D578G SEQ ID NOS: 374-375, D578K SEQID NOS: 376-377, D578M SEQ ID NOS: 378-379, D578N SEQ ID NOS: 380-381,D578S SEQ ID NOS: 382-383, D578T SEQ ID NOS: 384-385, D578W SEQ ID NOS:386-387, D578R/D732R SEQ ID NOS: 388-389, D578R/E742K SEQ ID NOS:390-391, D578R/D732R/E742K SEQ ID NOS: 392-393 E601K SEQ ID NOS:394-395, E602K SEQ ID NOS: 396-397, V607S SEQ ID NOS: 398-399, G603P SEQID NOS: 400-401, E681K SEQ ID NOS: 402-403, L682S SEQ ID NOS: 404-405,E694K SEQ ID NOS: 406-407, P701G SEQ ID NOS: 408-409, K702E SEQ ID NOS:410-411, E708K SEQ ID NOS: 412-413, D732R SEQ ID NOS: 414-415, D732C SEQID NOS: 416-417, D732F SEQ ID NOS: 418-419, D732G SEQ ID NOS: 420-421,D732H SEQ ID NOS: 422-423, D732K SEQ ID NOS: 424-425, D732N SEQ ID NOS:426-427, D732Q SEQ ID NOS: 428-429, D732S SEQ ID NOS: 430-431, D732T SEQID NOS: 432-433, D732R/E742K SEQ ID NOS: 434-435, E734K SEQ ID NOS:436-437, V737S SEQ ID NOS: 438-439, E742K SEQ ID NOS: 440-441, E742A SEQID NOS: 442-443, E742C SEQ ID NOS: 444-445, E742F SEQ ID NOS: 446-447,E742G SEQ ID NOS: 448-449, E742I SEQ ID NOS: 450-451, E742L SEQ ID NOS:452-453, E742M SEQ ID NOS: 454-455, E742P SEQ ID NOS: 456-457, E742T SEQID NOS: 458-459, E742V SEQ ID NOS: 460-461, E742W SEQ ID NOS: 462-463,E742Y SEQ ID NOS: 464-465, E745K SEQ ID NOS: 466-467, M765S SEQ ID NOS:468-469, E774K SEQ ID NOS: 470-471, L781S SEQ ID NOS: 472-473, A791F SEQID NOS: 474-475, E805K SEQ ID NOS: 476-477, L813S SEQ ID NOS: 478-479

The invention further includes mutant Taq polymerase having one or moreof the amino acid point substitution shown above and wherein theremainder of the Taq polymerase sequence is at least 70%, or at least75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 98%, or at least 99%, identical with wild type Taqpolymerase.

The invention further includes the nucleic acid sequences encoding anyof the above mutant Taq polymerases, including the correspondingsequences set forth in the Sequence Listing, and all degenerate nucleicacid sequences encoding the amino acid sequences of any of the mutantTaq polymerases shown above or set forth in the Sequence Listing; aswell as vectors incorporating such nucleic acid sequences and cellstransformed with such nucleic acid sequences and capable of expressingany of the above mutant Taq polymerases.

The invention further includes a composition or a kit comprising any ofthe above mutant Taq polymerases, the nucleic acid sequences encodingthem, or vectors incorporating such nucleic acid sequences. Theinvention also includes a process of amplifying a target nucleic acid,wherein any of the above mutant Taq polymerases are employed in areaction mixture designed to amplify a target nucleic acid, andsubjecting the reagent mixture to conditions for amplification of thetarget nucleic acid.

The above mutant Taq polymerases can amplify target DNA sequences moreeffectively than wild type, where the PCR is conducted in blood, plasma,or serum, including with sodium citrate as a preservative, or in anyreaction mixture with relatively high salt concentrations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows consolidated images of a number of gels, each representingthe results of PCR using one of the mutant Taq polymerases. Each suchmutant is indicated by a number, where the numbers correspond to themutation sites and sequences listed in Table 1, in the order shown inFIG. 1 , as follows:

708: E708K SEQ ID NOS: 412-413 732: D732R SEQ ID NOS: 414-415 734: E734KSEQ ID NOS: 436-437 737: V737S SEQ ID NOS: 438-439 742: E742K SEQ IDNOS: 440-441 745: E745K SEQ ID NOS: 466-467 765: M765S SEQ ID NOS:468-469 774: E774K SEQ ID NOS: 470-471 781: L781S SEQ ID NOS: 472-473791: A791F SEQ ID NOS: 474-475 805: E805K SEQ ID NOS: 476-477 813: L813SSEQ ID NOS: 478-479 534: Q534R SEQ ID NOS: 312-313 537: E537K SEQ IDNOS: 314-315 546: I546S SEQ ID NOS: 356-357 547: D547R SEQ ID NOS:358-359 551: D551R SEQ ID NOS: 362-363 553: I553S SEQ ID NOS: 366-367555: P555G SEQ ID NOS: 368-369 565: N565R SEQ ID NOS: 370-371 578: D578RSEQ ID NOS: 372-373 603: G603P SEQ ID NOS: 400-401 681: E681K SEQ IDNOS: 402-403 682: L682S SEQ ID NOS: 404-405 694: E694K SEQ ID NOS:406-407 701: P701G SEQ ID NOS: 408-409 702: K702E SEQ ID NOS: 410-411233: L233S SEQ ID NOS: 178-179 256: V256S SEQ ID NOS: 180-181 288: E288KSEQ ID NOS: 186-187 303: E303K SEQ ID NOS: 188-189 315: E315K SEQ IDNOS: 192-193 336: P336G SEQ ID NOS: 200-201 337: E337K SEQ ID NOS:202-203 351: L351S SEQ ID NOS: 208-209 373: P373G SEQ ID NOS: 216-217381: D381R SEQ ID NOS: 218-219 452: D452R SEQ ID NOS: 222-223 485: N485RSEQ ID NOS: 226-227 507: E507K SEQ ID NOS: 228-229 520: E520K SEQID NOS:268-269 521: A521F SEQ ID NOS: 310-311 76: E76K SEQ ID NOS: 26-27 91:D91R SEQ ID NOS: 28-29 104: D104R SEQ ID NOS: 32-33 111: L111S SEQ IDNOS: 34-35 186: T186I SEQ ID NOS: 36-37 189: E189K SEQ ID NOS: 38-39201: E201K SEQ ID NOS: 112-113 209: E209K SEQ ID NOS: 114-115 215: E215KSEQ ID NOS: 116-117 219: K219E SEQ ID NOS: 118-119 221: L221S SEQ IDNOS: 120-121 222: D222R SEQID NOS: 122-123 230: E230K SEQ ID NOS:124-125

and wherein for each gel, the PCR amplification products were subject togel electrophoresis separation following amplification of a targetnucleic acid having the sequence of SEQ ID NO: 1. There are six lanesfor each gel (as marked along the bottom of FIG. 1 ), where the two leftlane represent the results following PCR with the mutant Taq polymeraseat a concentration of 50 ng/μl; the next two lanes represent the resultswhere the mutant Taq polymerase was at a concentration of 5 ng/μl; andthe far right two lanes represent the results where the mutant Taqpolymerase was at a concentration of 0.5 ng/μl. There are two rows foreach gel, where the uppermost row represents the results following PCRwith the mutant Taq polymerase at a salt concentration of 110 mM KCl(though in the figure it is incorrectly labeled as “100 mM KCl”); andthe lowermost row represents the results following PCR with the mutantTaq polymerase at a salt concentration of 10 mM KCl (though in thefigure it is incorrectly labeled as “0 mM KCl”). The term “WT” refers towild type Taq polymerase.

FIG. 2 shows consolidated images of a number of gels, each representingthe results of PCR using one of the mutant Taq polymerases. Each suchmutant is indicated by a number, where the numbers correspond to themutation sites and sequences listed in Table I, in the order shown inFIG. 2 , as follows:

365: L365S SEQ ID NOS: 210-211 366: G366P SEQ ID NOS: 212-213 368: P368GSEQ ID NOS: 214-215 384: N384R SEQ ID NOS: 220-221 541: L541S SEQ IDNOS: 354-355 550: P550G SEQ ID NOS: 360-361 552: L552S SEQ ID NOS:364-365 601: E601K SEQ ID NOS: 394-395 602: E602K SEQ ID NOS: 396-397607: V6075 SEQ ID NOS: 398-399 101: E101K SEQ ID NOS: 30-31 260: K260ESEQ ID NOS: 182-183 261: R261D SEQ ID NOS: 184-185 309: F309A SEQ IDNOS: 190-191 320: D320R SEQ ID NOS: 194-195 328: R328D SEQ ID NOS:196-197 330: G330P SEQ ID NOS: 198-199 338: P338G SEQ ID NOS: 204-205342: L342S SEQ ID NOS: 206-207

Mutant Taq polymerases shown are those which had been shown to beeffective in high salt concentration PCR amplification. The lanes androws or each gel have the same mutant Taq polymerase and saltconcentrations (also incorrectly labeled as 100 mM KCl and 0 mM KCl,instead of 110 mM KCl and 10 mM KCl, respectively), as indicated in thesummary above for FIG. 1 .

FIG. 3 shows consolidated images of a number of gels, each representingthe results of PCR using one of the mutant Taq polymerases, where eachmutant is labeled the same way as the mutation sites and sequenceslisted in Table I. The lanes and rows or each gel have the same mutantTaq polymerase and salt concentrations (110 mM KCl and 10 mM KCl,respectively) as indicated in the summary above for FIG. 1 .

FIG. 4 shows consolidated images of a number of gels, each representingthe results of PCR using one of the mutant Taq polymerases, where eachmutant is labeled the same way as the mutation sites and sequenceslisted in Table I. The lanes and rows or each gel have the same mutantTaq polymerase and salt concentrations (110 mM KCl and 10 mM KCl,respectively) as indicated in the summary above for FIG. 1 .

FIG. 5 shows consolidated images of a number of gels, each representingthe results of PCR using one of the mutant Taq polymerases, where eachmutant is labeled the same way as the mutation sites and sequenceslisted in Table I. The lanes and rows or each gel have the same mutantTaq polymerase and salt concentrations (110 mM KCl and 10 mM KCl,respectively) as indicated in the summary above for FIG. 1 .

FIG. 6 shows consolidated images of a number of gels, each representingthe results of PCR using one of the mutant Taq polymerases, where eachmutant is labeled the same way as the mutation sites and sequenceslisted in Table I. The lanes and rows or each gel have the same mutantTaq polymerase and salt concentrations (110 mM KCl and 10 mM KCl,respectively) as indicated in the summary above for FIG. 1 .

SUMMARY DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NOS 4 and 5 are the respective amino acid and nucleotidesequences of histamine-tagged wild type Taq polymerase. The amino acidand DNA sequences of the various mutants listed in Table I above, areset forth in the sequence listing attached in the same order as in TableI, starting with the first amino acid sequence for E39K in Table I beingSEQ ID NO: 6 (a mutant Taq polymerase amino acid sequence).

Each mutant Taq polymerase amino acid sequence (which are the evennumbered sequences from SEQ ID NO: 6 to SEQ ID NO: 478) is immediatelyfollowed by its unique encoding sequence (which are the odd numberedsequences from SEQ ID NO: 7 to SEQ ID NO: 479).

DETAILED DESCRIPTION Definitions

The term “biologically active fragment” refers to any fragment,derivative, homolog or analog of a mutant Taq polymerase that possessesan in vivo or in vitro activity that is characteristic of thatbiomolecule. For example, mutant Taq polymerase can be characterized byvarious biological activities, including DNA binding activity,nucleotide polymerization activity, primer extension activity, stranddisplacement activity, reverse transcriptase activity, nick-initiatedpolymerase activity, 3′-5′ exonuclease (proofreading) activity,thermostability, ionic stability, accuracy, processivity, and the like.A “biologically active fragment” of a mutant Taq polymerase is anyfragment, derivative, homolog or analog that can catalyze thepolymerization of nucleotides (including homologs and analogs thereof)into a nucleic acid strand. In some embodiments, the biologically activefragment, derivative, homolog or analog of the mutant Taq polymerasepossesses 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% 95%, or98% or greater of the biological activity of the mutant Taq polymerasein any in vivo or in vitro assay of interest such as, for example, DNAbinding assays, nucleotide polymerization assays (which may betemplate-dependent or template-independent), primer extension assays,strand displacement assays, reverse transcriptase assays, proofreadingassays, accuracy assays, thermostabilty assays, ionic stability assaysand the like.

The biological activity of a polymerase fragment can be assayed bymeasuring any of: the primer extension activity in vitro of the fragmentunder defined reaction conditions; the polymerization activity in vitroof the fragment under defined reaction conditions; the thermostabilty invitro of the fragment under defined reaction conditions; the stabilityin vitro of the fragment under high ionic strength conditions; theaccuracy in vitro of the fragment under defined reaction conditions; theprocessivity in vitro of the fragment under defined reaction conditions;the strand displacement activity in vitro of the fragment under definedreaction conditions; the read-length activity in vitro of the fragmentunder defined reaction conditions; the strand bias activity in vitro ofthe fragment under defined reaction conditions; the proofreadingactivity in vitro of the fragment under defined reaction conditions; theoutput of an in vitro assay such as sequencing throughput or averageread length as performed by the polymerase fragment under definedreaction conditions; and, the output of a nucleotide polymerizationreaction in vitro such as raw accuracy of the polymerase fragment toincorporate correct nucleotides in the nucleotide polymerizationreaction under defined reaction conditions.

In some embodiments, a biologically active fragment can include any partof the DNA binding domain or any part of the catalytic domain of themutant Taq polymerase. In some embodiments, the biologically activefragment can optionally include any 25, 50, 75, 100, 150 or morecontiguous amino acid residues of the mutant Taq polymerase. Abiologically active fragment of a modified polymerase can include atleast 25 contiguous amino acid residues having at least 80%, 85%, 90%,95%, 98%, or 99% identity to any one or more of the even numberedsequences from SEQ ID NO: 4 to SEQ ID NO: 478. The invention alsoincludes the polynucleotides encoding any of the foregoing amino acidsequences (which are the coding portions of the odd numbered sequencesfrom SEQID NO: 5 to SEQID NO: 479, where each odd numberedpolynucleotide sequence encodes the previous even-numbered mutant Taqpolymerase).

Biologically active fragments can arise from post transcriptionalprocessing or from translation of alternatively spliced RNAs, oralternatively can be created through engineering, bulk synthesis, orother suitable manipulation. Biologically active fragments includefragments expressed in native or endogenous cells as well as those madein expression systems such as, for example, in bacterial, yeast, plant,insect or mammalian cells.

As used herein, the phrase “conservative amino acid substitution” or“conservative mutation” refers to the replacement of one amino acid byanother amino acid with a common property. A functional way to definecommon properties between individual amino acids is to analyze thenormalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz (1979) Principles of ProteinStructure, Springer-Verlag). According to such analyses, groups of aminoacids can be defined where amino acids within a group exchangepreferentially with each other, and therefore resemble each other mostin their impact on the overall protein structure (Schulz (1979) supra).Examples of amino acid groups defined in this manner can include: a“charged/polar group” including Glu, Asp, Asn, GIn, Lys, Arg, and His;an “aromatic or cyclic group” including Pro, Phe, Tyr, and Trp; and an“aliphatic group” including Gly, Ala, Val, Leu, lie, Met, Ser, Thr, andCys. Within each group, subgroups can also be identified. For example,the group of charged/polar amino acids can be sub-divided intosub-groups including: the “positively-charged sub-group” comprising Lys,Arg and His; the “negatively-charged sub-group” comprising Glu and Asp;and the “polar sub-group” comprising Asn and GIn. In another example,the aromatic or cyclic group can be sub-divided into sub-groupsincluding: the “nitrogen ring sub-group” comprising Pro, His, and Trp;and the “phenyl sub-group” comprising Phe and Tyr. In another furtherexample, the aliphatic group can be sub-divided into sub-groupsincluding: the “large aliphatic non-polar sub-group” comprising Val,Leu, and lie; the “aliphatic slightly-polar sub-group” comprising Met,Ser, Thr, and Cys; and the “small-residue sub-group” comprising Gly andAla. Examples of conservative mutations include amino acid substitutionsof amino acids within the sub-groups above, such as, but not limited to:Lys for Arg or vice versa, such that a positive charge can bemaintained; Glu for Asp or vice versa, such that a negative charge canbe maintained; Ser for Thr or vice versa, such that a free —OH can bemaintained; and GIn for Asn or vice versa, such that a free —NH2 can bemaintained. A “conservative variant” is a polypeptide that includes oneor more amino acids that have been substituted to replace one or moreamino acids of the reference polypeptide (for example, a polypeptidewhose sequence is disclosed in a publication or sequence database, orwhose sequence has been determined by nucleic acid sequencing) with anamino acid having common properties, e.g., belonging to the same aminoacid group or sub-group as delineated above.

When referring to a gene, “mutant” means the gene has at least one base(nucleotide) change, deletion, or insertion with respect to a native orwild type gene. The mutation (change, deletion, and/or insertion of oneor more nucleotides) can be in the coding region of the gene or can bein an intron, 3′ UTR, 5′ UTR, or promoter region. As nonlimitingexamples, a mutant gene can be a gene that has an insertion within thepromoter region that can either increase or decrease expression of thegene; can be a gene that has a deletion, resulting in production of anonfunctional protein, truncated protein, dominant negative protein, orno protein; or, can be a gene that has one or more point mutationsleading to a change in the amino acid of the encoded protein or resultsin aberrant splicing of the gene transcript.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism, like theTaq polymerase sequence, which has not been intentionally modified byhuman manipulation.

The terms “percent identity” or “homology” with respect to nucleic acidor polypeptide sequences are defined as the percentage of nucleotide oramino acid residues in the candidate sequence that are identical withthe known polypeptides, after aligning the sequences for maximum percentidentity and introducing gaps, if necessary, to achieve the maximumpercent homology. N-terminal or C-terminal insertion or deletions shallnot be construed as affecting homology. Homology or identity at thenucleotide or amino acid sequence level can be determined by BLAST(Basic Local Alignment Search Tool) analysis using the algorithmemployed by the programs blastp, blastn, blastx, tblastn, and tblastx(Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990),Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored forsequence similarity searching. The approach used by the BLAST program isto first consider similar segments, with and without gaps, between aquery sequence and a database sequence, then to evaluate the statisticalsignificance of all matches that are identified, and finally tosummarize only those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul (1994), Nature Genetics 6, 119-129.The search parameters for histogram, descriptions, alignments, expect(i.e., the statistical significance threshold for reporting matchesagainst database sequences), cutoff, matrix, and filter (low complexity)can be at the default settings. The default scoring matrix used byblastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff(1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended forquery sequences over 85 units in length (nucleotide bases or aminoacids).

Using the Mutant Taq Polymerase

In some embodiments, the invention relates to methods (and related kits,systems, apparatuses and compositions) for performing a nucleotidepolymerization reaction comprising or consisting of contacting a mutantTaq polymerase or a biologically active fragment thereof with a nucleicacid template in the presence of one or more nucleotides, andpolymerizing at least one of the one or more nucleotides using themodified polymerase or the biologically active fragment thereof. Themutant Taq polymerase or the biologically active fragment thereofincludes one or more amino acid modifications set forth in Table Irelative to wild type, and the mutant Taq polymerase or the biologicallyactive fragment thereof has an increased activity in a high ionicstrength solution, including in a solution up to 110 mM salt or KCl.

In some embodiments, the method can further include polymerizing atleast one nucleotide in a template-dependent fashion. In someembodiments, the polymerizing is performed under thermocyclingconditions. In some embodiments, the method can further includehybridizing a primer to the nucleic acid template prior to, during, orafter the contacting, and where the polymerizing includes polymerizingat least one nucleotide onto an end of the primer using the mutant Taqpolymerase or the biologically active fragment thereof. In someembodiments, the polymerizing is performed in the proximity of a sensorthat is capable of detecting the polymerization or the biologicallyactive fragment thereof. In some embodiments, the method can furtherinclude detecting a signal indicating the polymerization by the modifiedpolymerase or the biologically active fragment thereof using a sensor.In some embodiments, the sensor is an ISFET. In some embodiments, thesensor can include a detectable label or detectable reagent within thepolymerizing reaction.

In some embodiments, the method further includes determining theidentity of the one or more nucleotides polymerized by the modifiedpolymerase. In some embodiments, the method further includes determiningthe number of nucleotides polymerized by the modified polymerase. Insome embodiments, the polymerization occurs in the presence of a highionic strength solution of at least 110 mM salt. In some embodiments,the high ionic strength solution comprises KCl and/or NaCl.

In some embodiments, the invention relates to methods (and related kits,systems, apparatus and compositions) for detecting nucleotideincorporation comprising or consisting of performing a nucleotideincorporation reaction using a mutant Taq polymerase or a biologicallyactive fragment thereof, a nucleic acid template, and one or morenucleotide triphosphates; generating the nucleotide incorporation; anddetecting the nucleotide incorporation. Detecting nucleotideincorporation can occur via any appropriate means such as PAGE,fluorescence, dPCR quantitation, nucleotide by-product production (e.g.,hydrogen ion or pyrophosphate detection; suitable nucleotide by-productdetection systems include without limitation, next-generation sequencingplatforms such as Rain Dance, Roche 454, and Ion Torrent Systems)) ornucleotide extension product detection (e.g., optical detection ofextension products or detection of labelled nucleotide extensionproducts). In some embodiments, the methods (and related kits, systems,apparatus and compositions) for detecting nucleotide incorporationinclude or consist of detecting nucleotide incorporation using a mutantTaq polymerase or a biologically active fragment thereof.

In some embodiments, the invention relates to methods (and related kits,systems, apparatus and compositions) for amplifying a nucleic acid bycontacting it with a mutant Taq polymerase or a biologically activefragment thereof under suitable conditions for amplification of thenucleic acid; amplifying the nucleic acid using a polymerase chainreaction, emulsion polymerase chain reaction, isothermal amplificationreaction, recombinase polymerase amplification reaction, proximityligation amplification, rolling circle amplification or stranddisplacement amplification. The amplifying includes clonally amplifyingthe nucleic acid in solution, as well as clonally amplifying the nucleicacid on a solid support such as a nucleic acid bead, flow cell, nucleicacid array, or wells present on the surface of the solid support.

In some embodiments the method for amplifying a nucleic acid includesamplifying it under bridge PCR conditions. The bridge PCR conditionsinclude hybridizing one or more of the amplified nucleic acids to asolid support. The hybridized one or more amplified nucleic acids can beused as a template for further amplification.

In some embodiments, the disclosure generally relates to methods (andrelated kits, systems, apparatus and compositions) for synthesizing anucleic acid by incorporating at least one nucleotide onto the end of aprimer using a mutant Taq polymerase or a biologically active fragmentthereof. Optionally, the method further includes detecting incorporationof the at least one nucleotide onto the end of the primer. In someembodiments, the method further includes determining the identity of atleast one of the at least one nucleotide incorporated onto the end ofthe primer. In some embodiments, the method can include determining theidentity of all nucleotides incorporated onto the end of the primer. Insome embodiments, the method includes synthesizing the nucleic acid in atemplate-dependent manner. In some embodiments, the method can includesynthesizing the nucleic acid in solution, on a solid support, or in anemulsion (such as emPCR).

Making the Mutant Taq Polymerase

In some embodiments, in order to provide a mutant Taq polymerase whichcan withstand a salt concentration typically found in vivo, amino acidsubstitutions may be at one or more amino acids, 2 or more amino acids,3 or more amino acids or more, including where up to 30% of the totalnumber of amino acids of the wild type sequence are substituted.Embodiments of the mutant Taq polymerase may be anywhere from 70% to99.99% identical to the wild type. All embodiments of the mutant Taqpolymerase include one or more of the substitutions shown in Table I,and may also include substitutions, insertions or modifications to theremaining portions of the wild type sequence. In some embodiments, inorder to provide a mutant Taq polymerase which can withstand a saltconcentration typically found in vivo, several of the substitutionsshown in Table I may be included. These additional substitutions are notlimited to the combinations shown in Table I. For example, Table showsthe following combinations: E189K/E230K/E537K and E189K/E507K/E537K.These particular combinations, as well as other combinations in Table I,can be used with other substitutions in Table I, or with a mutant Taqpolymerase which includes substitutions, insertions or modifications toother portions of the wild type sequence.

The mutant Taq polymerases of the invention can be expressed in anysuitable host system, including a bacterial, yeast, fungal, baculovirus,plant or mammalian host cell. For bacterial host cells, suitablepromoters for directing transcription of the nucleic acid constructs ofthe present disclosure, include the promoters obtained from the E. colilac operon, Streptomyces coelicolor agarase gene (dagA), Bacillussubtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylasegene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter(DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80: 21-25).

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

For baculovirus expression, insect cell lines derived from Lepidopterans(moths and butterflies), such as Spodoptera frugiperda, are used ashost. Gene expression is under the control of a strong promoter, e.g.,pPolh.

Plant expression vectors are based on the Ti plasmid of Agrobacteriumtumefaciens, or on the tobacco mosaic virus (TMV), potato virus X, orthe cowpea mosaic virus. A commonly used constitutive promoter in plantexpression vectors is the cauliflower mosaic virus (CaMV) 35S promoter.

For mammalian expression, cultured mammalian cell lines such as theChinese hamster ovary (CHO), COS, including human cell lines such as HEKand HeLa may be used to produce the mutant Taq polymerase.

Examples of mammalian expression vectors include the adenoviral vectors,the pSV and the pCMV series of plasmid vectors, vaccinia and retroviralvectors, as well as baculovirus. The promoters for cytomegalovirus (CMV)and SV40 are commonly used in mammalian expression vectors to drive geneexpression. Non-viral promoters, such as the elongation factor (EF)-1promoter, are also known.

The control sequence for the expression may also be a suitabletranscription terminator sequence, that is, a sequence recognized by ahost cell to terminate transcription. The terminator sequence isoperably linked to the 3′ terminus of the nucleic acid sequence encodingthe polypeptide. Any terminator which is functional in the host cell ofchoice may be used.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase.

Terminators for insect, plant and mammalian host cells are also wellknown.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Signal peptides for other host cell systems are also wellknown.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the mutant Taq polymerase relative tothe growth of the host cell. Examples of regulatory systems are thosewhich cause the expression of the gene to be turned on or off inresponse to a chemical or physical stimulus, including the presence of aregulatory compound. In prokaryotic host cells, suitable regulatorysequences include the lac, tac, and trp operator systems. In yeast hostcells, suitable regulatory systems include, as examples, the ADH2 systemor GAL1 system. In filamentous fungi, suitable regulatory sequencesinclude the TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter. Regulatorysystems for other host cells are also well known.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the KRED polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Another embodiment includes a recombinant expression vector comprising apolynucleotide encoding an engineered mutant Taq polymerase or a variantthereof, and one or more expression regulating regions such as apromoter and a terminator, and a replication origin, depending on thetype of hosts into which they are to be introduced. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the mutant Taq polymerase at suchsites. Alternatively, the nucleic acid sequences of the mutant Taqpolymerase may be expressed by inserting the nucleic acid sequences or anucleic acid construct comprising the sequences into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the mutant Taqpolymerase polynucleotide sequence. The choice of the vector willtypically depend on the compatibility of the vector with the host cellinto which the vector is to be introduced. The vectors may be linear orclosed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector herein preferably contain one or more selectablemarkers, which permit easy selection of transformed cells. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers, whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol (Example 1) or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus. Selectable markers for insect, plant andmammalian cells are also well known.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). The integrational elements may be anysequence that is homologous with the target sequence in the genome ofthe host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori, or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAM31 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, ProcNatl Acad Sci. USA 75:1433).

More than one copy of a nucleic acid sequence of the mutant Taqpolymerase may be inserted into the host cell to increase production ofthe gene product. An increase in the copy number of the nucleic acidsequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

Expression vectors for the mutant Taq polymerase polynucleotide arecommercially available. Suitable commercial expression vectors includep3xFLAG™ expression vectors from Sigma-Aldrich Chemicals, St. Louis Mo.,which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

Suitable host cells for expression of a polynucleotide encoding themutant Taq polymerase polypeptide of the present disclosure, are wellknown in the art and include but are not limited to, bacterial cells,such as E. coli, Lactobacillus kefir, Lactobacillus brevis,Lactobacillus minor, Streptomyces and Salmonella typhimurium cells;fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae orPichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culturemediums and growth conditions for the above-described host cells arewell known in the art.

Polynucleotides for expression of the mutant Taq polymerase polypeptidemay be introduced into cells by various methods known in the art.Techniques include among others, electroporation, biolistic particlebombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion. Various methods for introducingpolynucleotides into cells are known to the skilled artisan.

Polynucleotides encoding the mutant Taq polymerase can be prepared bystandard solid-phase methods, according to known synthetic methods. Insome embodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides can be prepared by chemicalsynthesis using, e.g., the classical phosphoramidite method described byBeaucage et al., 1981, Tet Lett 22:1859-69, or the method described byMatthes et al., 1984, EMBO J. 3:801-05, e.g., as it is typicallypracticed in automated synthetic methods. According to thephosphoramidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors. In addition, essentially any nucleic acid can beobtained from any of a variety of commercial sources, such as TheMidland Certified Reagent Company, Midland, Tex., The Great AmericanGene Company, Ramona, Calif., ExpressGen Inc. Chicago, Ill., and OperonTechnologies Inc., Alameda, Calif.

Engineered mutant Taq polymerase expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the mutant Taq polymerasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions forpurification will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, andwill be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate themutant Taq polymerase. For affinity chromatography purification, anyantibody which specifically binds the mutant Taq polymerase polypeptidemay be used. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., may be immunizedby injection with a compound. The compound may be attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group. Various adjuvants maybe used to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacilli Calmette Guerin) andCorynebacterium parvum.

EXAMPLES

The wild type and mutant Taq polymerases were used in a PCR to amplifyan exemplary target nucleic acid having the sequence of SEQID NO: 1:

CAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGA;using a forward primer having SEQ ID NO: 2: CAGTGCTGCAATGATACC, and areverse primer having SEQ ID NO: 3: TCCTTGAGAGTTTTCGCC. The various KClconcentrations and various dilutions of the Taq polymerase (wild typeand mutant) were as set forth in the Brief Description of the Drawingsabove. The PCR was conducted using each of the mutant Taq polymerases,with a wild type as control, one at a time, and with the followingreagents and proportions in the PCR mixture:

-   -   4 μl of mutant (or wild type) Taq polymerase solution, wherein        the starting concentration of the Taq polymerase was 50 ng/μl,        was subject to 10-fold serial dilution in the experiments whose        results are shown in FIGS. 1-43 ; and it was subject to 2-fold        serial dilution in the experiments where the results are shown        in FIGS. 44-63 .    -   10× buffer 2 μl (wherein the 10× buffer consists of: 200 mM        Tris-HCl, 100 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgSO₄, 1% Triton®        X-100 pH 8.8@25° C.);    -   Primer Forward, SEQ ID NO: 2 (100 uM) 0.1 μl;    -   Primer Reverse, SEQ ID NO: 3 (100 uM) 0.1 μl;    -   dNTP (10 mM) 0.8 μl; and    -   pUC19 (1 ng/μl) 1 μl.    -   The salt, KCl, was added as needed to achieve the salt        concentrations indicated in the FIGS. 1-63 for the various        experimental runs. The reaction mixture was made up to a total        volume of 20 μl by adding distilled water.

After the PCR amplification of SEQ ID NO: 1, the amplicons in thereaction mixture were separated using gel electrophoresis, under thefollowing conditions: the gel used was 1.2% Agarose (Agarose LE,Goldbio.com, cat: A-2Q1-1000); the buffer was: 1× TBE buffer, (ResearchProducts International, cat: T22020-10.0); the gel size was 12×14 cm;and the electrophoresis was run at 200V, for 20 minutes.

Example I

The consolidated results from the electrophoresis are shown in FIGS. 1to 6 , which are consolidated images of the individual gels for some ofthe mutant Taq polymerases listed in Table I.

The results of the experiments conducted herein (as in FIGS. 1 to 6 )show that many of the mutant Taq polymerases in Table I are notsubstantially inhibited under salt concentrations up to 110 mM KCl.Nearly every one of the mutant Taq polymerases in FIGS. 1 to 6 showamplification of the target sequence (dark spots) at lowerconcentrations of the mutant Taq polymerases and/or at higher saltconcentration than did the wild type (“WT” in FIGS. 1-6 ). These mutantTaq polymerases and biologically active fragments thereof have beenshown to be effective in salt concentrations typical in body fluids,such as blood, serum and plasma, even if they are preserved using sodiumcitrate.

Example II

The mutant taq polymerases listed in Table I but not shown in FIGS. 1 to6 are used in PCR to amplify an exemplary target nucleic acid such asthe sequence of SEQ ID NO: 1. The various KCl concentrations and variousdilutions of the mutant Taq polymerases are as set forth in the BriefDescription of the Drawings above. The PCR is conducted using each ofthe mutant Taq polymerases, with a wild type as control, one at a time,and with reagents and proportions in the PCR mixture as in Example I.The experiments show that these mutant Taq polymerases are also notsubstantially inhibited under salt concentrations up to 110 mM KCl.

The osmolarity for blood, serum, plasma and even sodium citratepreserved blood has a lower osmolarity than 110 mM KCl, so it isexpected the mutant Taq polymerases will not be inhibited by thepresence in the sample of blood, serum, plasma, or other body fluids,even if they are preserved using sodium citrate.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “including”, containing”, etc. are to beread expansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. It is also noted thatas used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference, and the plural include singularforms, unless the context clearly dictates otherwise. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intent in the use of such terms andexpressions to exclude any equivalent of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention as claimed.Thus, it will be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

1-20. (canceled)
 21. A mutant Taq polymerase which is active in a wholeblood, plasma or serum, or in a PCR mixture which includes KCl at aconcentration of 10 to 110 mM, comprising one of the following aminoacid mutations preceding a comma, at the positions indicated in eachamino acid sequence and as fully shown in an adjacent even-numberedsequence identification number preceding a comma but wherein each saidamino acid sequence does not include the 6-membered histidine tag at itsC-terminus and the six immediately preceding Glycine and Serine aminoacids shown in each said amino acid sequence, and wherein a DNA encodingeach said amino acid sequence is the odd-numbered sequenceidentification number following each said even-numbered sequenceidentification number preceding a comma: V737S SEQ ID NOS: 438-439;E745K SEQ ID NOS: 466-467; M765S SEQ ID NOS: 468-469; E774K SEQ ID NOS:470-471; L781S SEQ ID NOS: 472-473; A791F SEQ ID NOS: 474-475; E805K SEQID NOS: 476-477; L813S SEQ ID NOS: 478-479; Q534R SEQ ID NOS: 312-313;E537K SEQ ID NOS: 314-315; I546S SEQ ID NOS: 356-357; and D547R SEQ IDNOS: 358-359.
 22. The mutant Taq polymerase of claim 21 wherein the PCRmixture further includes sodium.
 23. A mutant Taq polymerase which isactive in a whole blood, plasma or serum, or in a PCR mixture whichincludes KCl at a concentration of 10 to 110 mM, comprising one of thefollowing amino acid mutations preceding a comma, at the positionsindicated in each amino acid sequence and as fully shown in an adjacenteven-numbered sequence identification number preceding a comma butwherein each said amino acid sequence does not include the 6-memberedhistidine tag at its C-terminus and the six immediately precedingGlycine and Serine amino acids shown in each said amino acid sequence,and wherein a DNA encoding each said amino acid sequence is theodd-numbered sequence identification number following each saideven-numbered sequence identification number preceding a comma: I553SSEQ ID NOS: 366-367; P555G SEQ ID NOS: 368-369; N565R SEQ ID NOS:370-371; D578R SEQ ID NOS: 372-373; G603P SEQ ID NOS: 400-401; E681K SEQID NOS: 402-403; L682S SEQ ID NOS: 404-405; P701G SEQ ID NOS: 408-409;K702E SEQ ID NOS: 410-411; L233S SEQ ID NOS: 178-179; V256S SEQ ID NOS:180-181; E288K SEQ ID NOS: 186-187; E303K SEQ ID NOS: 188-189; P336G SEQID NOS: 200-201; E337K SEQ ID NOS: 202-203; L351S SEQ ID NOS: 208-209;P373G SEQ ID NOS: 216-217; D381R SEQ ID NOS: 218-219; and D452R SEQ IDNOS: 222-223.
 24. The mutant Taq polymerase of claim 23 wherein the PCRmixture further includes sodium.
 25. A mutant Taq polymerase which isactive in a whole blood, plasma or serum, or in a PCR mixture whichincludes KCl at a concentration of 10 to 110 mM, comprising one of thefollowing amino acid mutations preceding a comma, at the positionsindicated in each amino acid sequence and as fully shown in an adjacenteven-numbered sequence identification number preceding a comma butwherein each said amino acid sequence does not include the 6-memberedhistidine tag at its C-terminus and the six immediately precedingGlycine and Serine amino acids shown in each said amino acid sequence,and wherein a DNA encoding each said amino acid sequence is theodd-numbered sequence identification number following each saideven-numbered sequence identification number preceding a comma: E520KSEQ ID NOS: 268-269; A521F SEQ ID NOS: 310-311; D91R SEQ ID NOS: 28-29;D104R SEQ ID NOS: 32-33; L11 S SEQ ID NOS: 34-35; T186I SEQ ID NOS:36-37; E201K SEQ ID NOS: 112-113; E215K SEQ ID NOS: 116-117; K219E SEQID NOS: 118-119; L221S SEQ ID NOS: 120-121; D222R SEQ ID NOS: 122-123;L365S SEQ ID NOS: 210-211; G366P SEQ ID NOS: 212-213; P368G SEQ ID NOS:214-215; N384R SEQ ID NOS: 220-221; L541S SEQ ID NOS: 354-355; P550G SEQID NOS: 360-361; and L552S SEQ ID NOS: 364-365.
 26. The mutant Taqpolymerase of claim 25 wherein the PCR mixture further includes sodium.